ATR inhibitors and gemcitabine synergize to induce cell death in TP53-mutant, ATRX-wildtype uterine leiomyosarcoma
Megan E. Doherty, Minnie Malik, Yuka Otsuka, Nicholas W. Bateman, John I. Risinger, Nicholas Pyskir, Thomas P. Conrads, Kathleen M. Darcy, G Larry. Maxwell, Satishkumar Ranganathan. Ganakammal, Clifton L. Dalgard, Matthew D. Wilkerson, Gauthaman Sukumar, Joseph P. LaMorte, Bridget A. Finniff, Robert L. Kortum, William H. CatherinoAbstract
Inactivating mutations in TP53 and ATRX are key alterations defining the genomic landscape of uterine leiomyosarcoma (uLMS), creating chronic replication stress and increasing dependency on Ataxia telangiectasia and Rad3-related kinase (ATR) signaling for survival. ATR-CHK1 signaling limits the efficacy of standard-of-care gemcitabine by facilitating the stabilization of stalled replication forks and preventing lethal DNA strand breaks in checkpoint-deficient cells. Here, we show that ATR inhibitors (elimusertib, ceralasertib, berzosertib) preferentially synergize with gemcitabine over docetaxel or doxorubicin, across a panel of TP53-mutant/ATRX-wildtype uLMS models. When simultaneously administered, gemcitabine/elimusertib demonstrated enhanced synergy and efficacy compared to standard-of-care gemcitabine/docetaxel. This combination benefit was absent in a TP53-wildtype uterine sarcoma control line, highlighting TP53 mutation status as a critical predictive biomarker. Combined gemcitabine/ATRi treatment demonstrated reciprocal potency enhancement at low-nanomolar concentrations and significantly increased apoptotic populations compared to monotherapy. Gemcitabine monotherapy induced CHK1 (S345) phosphorylation and a corresponding S-phase arrest. Elimusertib abrogated this checkpoint, transforming protective S-phase arrest into a cytotoxic response characterized by a marked increase in the sub-G1 population. This transition was driven by the inhibition of gemcitabine-induced CHK1 phosphorylation, resulting in marked accumulation of unrepaired DNA damage (γH2AX) and the induction of apoptosis as evidenced by cleaved caspase-3 expression. These findings demonstrate that elimusertib strongly potentiates gemcitabine-induced damage to kill TP53-mutant uLMS in vitro, outperforming standard gemcitabine/docetaxel when concurrently administered. This combination strategy broadens the clinical utility of ATR inhibitors to uLMS harboring the prevalent TP53-mutant/ATRX-wildtype genotype and supports the clinical evaluation of gemcitabine/ATRi combinations as a novel therapy for uLMS.